Our long-term objectives are to develop lower-cost BMP (bone morphogenetic protein)-impregnated SBGSs (synthetic bone graft substitutes) for use in treating orthopaedic injuries and to elucidate the mechanism(s) of action of cBBP (cyclic BMP-binding peptide)/spp-18.5/spp-24. Recently, Urist's "native BMP/NCP" was found to be spp-18.5, an 18.5 kD degradation product of spp-24 (secreted phosphoprotein- 24 kD). spp-18.5 contains a cyclic 19-amino acid residue sequence designated cBBP that is similar to the cyclic TRH-1 (TGF-beta receptor homology-1) domains of fetuin and the receptor itself. cBBP binds rhBMP-2 (recombinant human BMP-2) in vitro, increases its tissue retention time in vivo, and increases the rate/magnitude of ectopic and calvarial bone formation in vivo, spp-18.5 (and, by extension, cBBP) is probably the matrix component that Wozney hypothesized contributed slow-release properties to rhBMP-2, thus reducing the amount of rhBMP-2 required to induce cartilage and bone. We hypothesize that cBBP will enhance down-stream BMP-2-mediated bone-specific and regulatory gene expression and bone formation in vitro and bone healing in vivo in SBGSs. Specific objective 1 is to probe the mechanism of action of cBBP by determining its effects on the rate and magnitude of rhBMP-2-mediated bone-specific and regulatory gene expression and bone formation in vitro in a SBGS consisting of BMSCs (bone marrow stromal cells) precultured in HA (hydroxyapatite) scaffolds by real time RT-PCR, ELISA and biochemical assays, microscopy and histology. Specific objective 2 is to validate the efficacy of cBBP in BMP-impregnated SBGSs by determining its effects on the rate and magnitude of BMP-2-mediated healing in vivo in a rabbit critical-sized calvarial defect repaired with a BMSC/HA SBGS by densitometry, microCT scanning, and quantitative static and dynamic bone histomorphometry as part of developing a cheaper BMP-impregnated SBGS. About 6 million fractures are treated annually in the U.S. Impaired bone healing occurs in 5%-10% of patients. This is a significant problem in orthopaedic reconstructive surgery, where many patients undergo repeated operative procedures that can result in suboptimal outcomes. BMPs promote fracture healing, but their use is limited by high cost, rapid diffusion, and short retention times. Improving the retention time of BMP in SBGSs could reduce medical care costs, reduce healing time, and improve clinical outcomes. [unreadable] [unreadable] [unreadable]